Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.10.026

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the prese

Full text of DOI:10.1016/j.bmcl.2018.10.026

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new potent protein arginine methyltransferase 5 (PRMT5)
inhibitors by assembly of key pharmacophores from known inhibitors
⁎
⁎
Kongkai Zhua,b, Jia-Li Songa, Hong-Rui Taoa, Zhi-Qiang Chenga, Cheng-Shi Jianga, , Hua Zhanga,
a
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a
promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been
put into phase I clinical trial. In the present study, a series of candidate molecules were designed by com-
bining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of
compound 4b14 revealed an IC50 of 2.71 μM, exhibiting high selectivity over PRMT1 and PRMT4 (> 70-fold
selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and
lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant
cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine di-
methylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interac-
tions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This
scaffold provides a new chemical template for further development of better lead compounds targeting
PRMT5.
Pharmacophore combination
Isoquinoline
PRMT5 inhibitor
Molecular docking
Molecular dynamics simulation
Protein methylation catalyzed by protein arginine methyl-
transferases (PRMTs) on arginine residues could influence many key
biological processes.1 As a member of the PRMT family, PRMT5
modulates the biological functions of its substrate proteins by sym-
metrically transferring up to two methyls to arginine residues.2 Me-
thylation mediated by PRMT5 is essential to maintain homeostasis in
normal cells. However, increasing studies indicate that PRMT5 is also
involved in cell growth and survival pathways that promote tumor-
igenesis and cancer development.3–14 Specifically, PRMT5 is fre-
quently overexpressed in diverse human cancers and its increased
activity has been evidenced to promote cell transformation.15 Besides,
PRMT5 was reported to be a critical in vitro and in vivo regulator of
breast cancer stem cell proliferation and self-renewal.4 Also, it was
demonstrated to control melanoma growth mediated by SHARPIN, the
latter being an adaptor for the linear ubiquitin chain assembly com-
plex.5 In multiple myeloma (MM), PRMT5 has been reported to be a
druggable target, as it is overexpressed in patient MM cells and has a
close relationship with prognostic relevance.6 All of these studies in-
dicate that PRMT5 is a promising target for anticancer drug devel-
opment.
reported PRMT5 inhibitors, as summarized in Fig. 1, can be classified
into two classes – SAM analogues and non-SAM analogues. Researchers
have made their focus on the development of non-SAM analogues. As
can be seen from the fact that except Sinefungin,20 DS-437,21
A9145C,20 compound 1,22 LLY-28325 and JNJ-64619178,26 all the
reported PRMT5 inhibitors belong to non-SAM analogues. Among them,
GSK-3326595 exhibited the most potent inhibitory activity at enzy-
matic level, with an IC50 of 6.2 nM.28 In phase I clinical trial, dose es-
calation study of this compound in solid tumors and non-Hodgkin's
lymphoma is undergoing. Its analogue EPZ0156665 displayed effective
anti-tumor activity in mantle cell lymphoma (MCL) xenograft models
and has been used as a tool compound to investigate the oncogenic role
of PRMT5. The Co-crystal structure of EPZ015666 with PRMT5:MEP50
complex (PDB code: 4X61)9 indicated that this series of compounds
occupied the substrate binding pocket of PRMT5.
Given the therapeutic potential of PRMT5 in cancer treatment and
its limited inhibitors, there is an urgent need to discover more in-
hibitory molecules. In this study, we reported the design, synthesis and
biological evaluation of a new potent PRMT5 inhibitor (4b14) with
high selectivity over other two key members of PRMTs (PRMT1 and
PRMT4). A panel of leukemia and lymphoma cells, including MV4-11,
Pfeiffer, SU-DHL-4 and KARPAS-422, were found to be sensitive to this
Like most promising therapeutic targets, great efforts have been
made to develop inhibitors against PRMT5.9,16–27 The previously
⁎
Corresponding authors.
E-mail addresses: jiangchengshi-20@163.com (C.-S. Jiang), bio_zhangh@ujn.edu.cn (H. Zhang).
https://doi.org/10.1016/j.bmcl.2018.10.026
Received 26 August 2018; Received in revised form 16 October 2018; Accepted 17 October 2018
0960-894X/©2018PublishedbyElsevierLtd.
Pleasecitethisarticleas:Zhu,K.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2018.10.026

K. Zhu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Reported PRMT5 inhibitors.
Fig. 2. Design strategy of compound 4a, 4b, and 4c. (A) Chemical structures of 17 and GSK-3326595. (B) Superimposition of binding modes of 17 and GSK-
3326595.
compound with EC50 values lower than 16.0 μM. Flow cytometric
analysis demonstrated that 4b14 showed obvious cell cycle arrest and
apoptosis-inducing effects. In SU-DHL-4 and KARPAS-422 cells, 4b14
reduced the cellular symmetric arginine dimethylation levels of SmD3,
a protein that has been popularly used to characterize the cellular
biochemical activity of PRMT5.29,30 The binding mode of 4b14 with
PRMT5 was probed by molecular docking and further validated by
molecular dynamics simulation. Affinity binding profiling analysis in-
dicated that hydrophobic interactions, π-π stacking and cation-π ac-
tions made the major contributions to the overall binding affinity.
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Scheme 1. Synthesis of 4a–4c. Reagents and condi-
tions: a) triethylamine, dichloromethane, 0 °C to r.t.,
overnight, 62–84%; b) 1,2,3,4-tetrahydroisoquino-
line, triethylamine, acetonitrile, microwave heating
100 °C, 10 min, 66%-83%.
Scheme 2. Synthesis of 4b1–4b20. Reagents and
conditions: a) triethylamine, dichloromethane, 0 °C
to r.t., overnight, 57%–85%; b) 1,2,3,4-tetra-
hydroisoquinoline, triethylamine, acetonitrile, mi-
crowave heating 100 °C, 10 min, 42%–79%.
Design and synthesis PRMT5 inhibitors
withdrawing group at 3-position had decreased activity or no obvious
inhibition on PRMT5. Since compounds 4b4 and 4b9 exhibited in-
creased activity, analogue 4b14 with 3-Br and 2-CN was next prepared.
As expected, 4b14 showed the most potent activity with an IC50 value
Compound 17 and GSK-3326595 were previously reported PRMT5
inhibitors. While 17 was identified and proposed to occupy the SAM
binding site in our recent work,16 GSK-3326595 was confirmed as a
substrate binding site inhibitor.9 A detailed analysis of their binding
modes allowed the identification of key pharmacophores in each
compound, and the following anticipation of the merging of these
fragments including tetrahydroiso-quinoline (blue color) and methyl 2-
acetamidobenzoate (red color). In order to investigate the optimal
distance between both fragments for desired activity, three alkyl linker
(black color) was first rationally exploited to yield the new potential
PRMT5 inhibitors 4a–4c (Fig. 2).
of 2.71
0.21 µM, being 11.7-fold as active as the initial hit 4b.
Further introduction of substituents on the 4-position of benzene
(4b15–4b17) did not have positive effects on the PRMT5 inhibitory
activity. In addition, replacement of the benzene ring with bulky
naphthalene or quinolone moieties also led to the loss of activity.
Selectivity test for 4b14
The synthetic route for 4a–4c was shown in Scheme 1. Briefly, the
intermediates 3a–3c were prepared with good yields of 62%–84%
through acylation reaction of methyl 2-aminobenzoate with corre-
sponding acyl chlorides 2a–2c. Then 3a–3c were reacted with 1,2,3,4-
tetrahydroisoquinoline under microwave condition to give corre-
sponding target compounds 4a–4c in 66%–85% yields. The three
compounds were then evaluated for PRMT5 inhibitory activity at en-
zymatic level. To investigate the substituent effect of phenylamine
fragment, twenty analogues of 4b were synthesized. Scheme 2 showed
the preparation of analogues 4b1–4b20 (Table 1), from the staring
materials 2b and 5b1-5b20 in 33%-58% overall yields, which was
achieved according to the same protocol as that of 4a–4c.
In enzyme-inhibitory assays using PRMT1 and PRMT4, compound
4b14 was assessed for its activity against the two enzymes to confirm its
selectivity. As shown in Fig. S1 (Supporting information), the IC50 va-
lues of 4b14 against PRMT1 and PRMT4 were both above 200 μM,
which indicated that 4b14 was a selective inhibitor of PRMT5 over
PRMT1 and PRMT4.
Mechanism of action and binding mode analysis
The direct binding of compound 4b14 to PRMT5 was confirmed by
surface plasmon resonance (SPR) assay with a KD (equilibrium dis-
sociation constant) value of 10.8
3.2 μM (Fig. 3A). The mechanism
PRMT5 inhibitory activity evaluation and structure-activity
relationship analysis
of action (MOA) of 4b14 was assessed by determining the IC50 values in
the presence of various concentrations of SAM and peptide. As shown in
Fig. 3B, 4b14 displayed competitive inhibition with peptide while
noncompetitive with SAM. This results indicated that 4b14 was a
substrate binding site inhibitor of PRMT5. To probe the probable
binding mode of 4b14 with PRMT5, molecular docking and molecular
dynamics simulation were performed. The putative binding mode and
the detailed interactions between 4b14 and PRMT5 were shown in
Fig. 3C and D, respectively. As indicated in Fig. 3D, although there are a
few polar interactions, the most are from hydrophobic, π-π stacking and
cation-π interactions as expected.
Radiometric-based scintillation proximity assay (SPA) was used to
detect the inhibitory activity of the synthesized 4a–4c against PRMT5.
The bioassay results showed that compound 4b displayed better PRMT5
inhibitory activity (IC50 = 31.62
2.15 µM) than 4a and 4c, in-
dicating that propionyl group (e(CH2)2COe) is the superior linker for
the improved activity.
To expand the substituent/fragment diversity and investigate elec-
tronic or steric effect on improving the PRMT5 inhibitory activity,
compound 4b was next used as a template based on the above bioassay
result. The introduction of different substituents into phenylamine
moiety or replacement of phenylamine moiety by other fragments were
carried out with chemical agents in hand, which resulted into the
production of 4b1–4b20. Among the compounds (4b1–4b4) with ortho-
substituents, 4b4 with 2-CN showed slightly better activity
Based on this binding mode, 100 ns molecular dynamics (MD) si-
mulation were performed to further validate the putative binding mode
and calculate the binding free energy. As shown in Fig. S2 (Supporting
information), the interactions between 4b14 and PRMT5 were found to
be stable during the MD simulation, by analyzing the root mean square
deviation (RMSD) values of PRMT5 (heavy atoms Cα, C, N) and 4b14,
respectively. Then the binding free energy was obtained by MM/PBSA
method to be −36.38 kcal/mol, which was well in accordance with its
enzymatic inhibition activity.
(IC50 = 25.55 1.67 µM) compared with 4b. From the assay results of
4b6–4b13, compound 4b9 with 3-Br showed the best activity
(IC50 = 18.12 0.71 µM), while others with more bulky or electron-
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Table 1
Table 1 (continued)
Inhibition of PRMT5 by series of compounds 4.
a
Compound
IC50
SDb (µM)
Compound
IC50
SDb (µM)
a
n
R
n
1
R
4b16
4b17
2
> 100
> 100
4a
60.02
31.62
87.89
> 100
1.44
2.15
4.27
2
4b
4c
2
3
2
4b18
2
72.65
3.21
4b19
4b20
2
2
> 100
> 100
4b1
4b2
2
> 100
Control (SAH)
0.56
0.03
4b3
4b4
2
2
> 100
25.55
8 (EPZ015666)
0.047
0.009
a
IC50: half maximal inhibitory concentration.
SD: standard deviation.
1.67
b
4b5
4b6
4b7
4b8
4b9
4b10
2
2
2
2
2
2
> 100
> 100
45.87
42.13
18.12
51.95
Anti-proliferative activity and cellular symmetric dimethylation
effects of 4b14
One leukemia (MV4-11) and three lymphoma (Pfeiffer, SU-DHL-4
and KARPAS-422) cell lines were used to measure the anti-proliferative
activity of 4b14. As shown in Fig. 4A, 4b14 showed a dose-dependent
inhibition against the four types of cells with 4-day IC50 values ranging
from 9.14 μM to 16.42 μM.
3.14
2.97
0.71
2.55
The effects of 4b14 and EPZ015666 (positive control) on cellular
symmetric arginine dimethylation in KARPAS-422 cells (Fig. 4B and
Fig. S3) were also tested by immunoblot using symmetric dimethyl
arginine (SDMA) antibody. In agreement with EPZ015666, treatment
of 4b14 on KARPAS-422 cells led to a concentration-dependent de-
crease in the level of symmetric arginine dimethylation of PRMT5
substrate SmD3, while in SU-DHL-4 cells this effect was not so obvious.
The assay results demonstrated that 4b14 could target PRMT5 in a
cellular context.
4b11
4b12
4b13
4b14
2
2
2
2
62.95
69.87
> 100
2.71
2.55
2.54
Inhibition of cell cycle progression and induction of cell apoptosis
PRMT5 inhibitors could result in the inhibition of cell proliferation
and/or induction of apoptosis.31 Thus, we investigated whether 4b14
could affect the progression of human lymphoma cancer KARPAS-422
cells through the cell cycle. As shown in Fig. 5A, the control cells dis-
played a characteristic distribution of cells in different phases of the cell
cycle. In contrast, compound 4b14 arrested the cell cycle in the G1
phase in a concentration-dependent manner, which was indicative of
the cellular engagement of 4b14 on targeting PRMT5.
0.21
4b15
2
> 100
To confirm the capacity of 4b14 to induce apoptosis, KARPAS-422
cells were treated with the compound for 48 h. As can be seen in
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Fig. 3. Mechanism of action and binding mode prediction of 4b14. (A) Binding of 4b14 to PRMT5 was confirmed by SPR. (B) Mechanism of action (MOA) of 4b14.
MOA was assessed by determining the IC50 values in the presence of various concentrations of peptide and SAM. Experiments were performed in triplicate. (C) The
proposed binding mode of 4b14 with PRMT5. (D) The detailed interactions between 4b14 and PRMT5.
Fig. 4. Antiproliferative effect and arginine me-
thylation alterations upon treatment with 4b14
in cell-based assays. (A) Treatment of MV4-11,
Pfeiffer, SU-DHL-4 and KARPAS-422 cells with
4b14 inhibited cell proliferation. Results shown
are mean
SD of three replicates. (B)
Treatment with 4b14 for 72 h dose-dependently
inhibited the methyltransferase activity of
PRMT5 in KARPAS-422 cells. SDMA levels were
detected by Western blotting.
Fig. 5B, compared to the control group, 4b14 was capable of inducing
apoptosis in a dose-dependent manner. After 48 h, the maximum
apoptotic rate of ∼20% was achieved at the concentration of 16.0 µM.
In conclusion, new PRMT5 inhibitors were successfully discovered
in the current study by combination of key pharmacophores from the
reported PRMT5 inhibitors. Among all the analogues, compound 4b14
showed the most potent PRMT5 inhibitory activity with an IC50 value of
below 16.0 μM. Flow cytometric analysis demonstrated that 4b14 ex-
hibited obvious cell cycle arrest and apoptosis-inducing effects. In SU-
DHL-4 and KARPAS-422 cells, 4b14 reduced the cellular symmetric
arginine dimethylation levels of SmD3, a protein used to characterize
the cellular biochemical activity of PRMT5. Besides, the binding mode
of 4b14 with PRMT5 was probed by molecular docking and further
validated by molecular dynamics simulation. Affinity binding profiling
analysis indicated that hydrophobic interaction contributed mainly to
the overall binding affinity. This compound provides a new scaffold for
further PRMT5 inhibitor development.
2.71
0.21 µM. Besides, this compound also displayed moderate anti-
proliferative activity against a panel of leukemia (MV4-11) and lym-
phoma (Pfeiffer, SU-DHL-4 and KARPAS-422) cells, with IC50 values all
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Fig. 5. Cell cycle progression and of cell apoptosis analysis. (A) Treatment with 4b14 arrested KARPAS-422 cells at G1 phase at 48 h. (B) Treatment with 4b14
induced apoptosis of KARPAS-422 cells at 48 h.
Acknowledgements
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